Solvent tolerant microorganisms and methods of isolation

ABSTRACT

Enterococcus  bacteria having enhanced tolerance to butanols have been isolated. The bacteria are useful for the fermentive production of butanol. New methods for the isolation of butanol tolerant  Enterococcus  are also provided.

This application claims the benefit of U.S. Provisional Application No. 60/798,417, filed May 5, 2006.

FIELD OF THE INVENTION

The invention relates to the field of industrial microbiology. Specifically, microorganisms have been isolated that demonstrate high tolerance to alcohols, particularly butanols.

BACKGROUND OF THE INVENTION

Butanol is an important industrial chemical, useful as a fuel additive, as a feedstock chemical in the plastics industry, and as a foodgrade extractant in the food and flavor industry. Each year 10 to 12 billion pounds of butanol are produced by petrochemical means and the need for this commodity chemical will likely increase.

Methods for the chemical synthesis of butanols are known. For example, 1-butanol may be produced using the Oxo process, the Reppe process, or the hydrogenation of crotonaldehyde (Ullmann's Encyclopedia of Industrial Chemistry, 6^(th) edition, 2003, Wiley-VCHVerlag GmbH and Co., Weinheim, Germany, Vol. 5, pp. 716-719). 2-Butanol may be produced using n-butene hydration (Ullmann's Encyclopedia of Industrial Chemistry, 6^(th) edition, 2003, Wiley-VCHVerlag GmbH and Co., Weinheim, Germany, Vol. 5, pp. 716-719). Additionally, isobutanol may be produced using Oxo synthesis, catalytic hydrogenation of carbon monoxide (Ullmann's Encyclopedia of Industrial Chemistry, 6^(th) edition, 2003, Wiley-VCHVerlag GmbH and Co., Weinheim, Germany, Vol. 5, pp. 716-719) or Guerbet condensation of methanol with n-propanol (Carlini et al., J. Molec. Catal. A:Chem. 220:215-220 (2004)). These processes use starting materials derived from petrochemicals, are generally expensive, and are not environmentally friendly.

Methods of producing butanol by fermentation are also known, where the most popular process produces a mixture of acetone, 1-butanol and ethanol and is referred to as the ABE processes (Blaschek et al., U.S. Pat. No. 6,358,717). Acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum is one of the oldest known industrial fermentations, and the pathways and genes responsible for the production of these solvents have been reported (Girbal et al., Trends in Biotechnology 16:11-16 (1998)). Additionally, recombinant microbial production hosts expressing a 1-butanol biosynthetic pathway (Donaldson et al., copending and commonly owned U.S. patent application Ser. No. 11/527,995), a 2-butanol biosynthetic pathway (Donaldson et al., copending and commonly owned U.S. Patent Application No. 60/796,816), and an isobutanol biosynthetic pathway (Maggio-Hall et al., copending and commonly owned U.S. patent application Ser. No. 11/586,315) have been described. However, biological production of butanols is believed to be limited by butanol toxicity to the host microorganism used in the fermentation.

Strains of Clostridium that are tolerant to 1-butanol have been isolated by chemical mutagenesis (Jain et al. U.S. Pat. No. 5,192,673; and Blaschek et al. U.S. Pat. No. 6,358,717), overexpression of certain classes of genes such as those that express stress response proteins (Papoutsakis et al. U.S. Pat. No. 6,960,465; and Tomas et al., Appl. Environ. Microbiol. 69(8):4951-4965 (2003)), and by serial enrichment (Quratulain et al., Folia Microbiologica (Prague) 40(5):467-471 (1995); and Soucaille et al., Current Microbiology 14(5):295-299 (1987)). Desmond et al. (Appl. Environ. Microbiol. 70(10):5929-5936 (2004)) report that overexpression of GroESL, a stress response protein, in Lactococcus lactis and Lactobacillus paracasei produced strains that were able to grow in the presence of 0.5% volume/volume (v/v) [0.4% weight/volume (w/v)] 1-butanol. Additionally, the isolation of 1-butanol tolerant strains from estuary sediment (Sardessai et al., Current Science 82(6):622-623 (2002)) and from activated sludge (Bieszkiewicz et al., Acta Microbiologica Polonica 36(3):259-265 (1987)) have been described. However, for most microorganisms described in the art, growth is totally inhibited at a concentration of less than 2.0% w/v 1-butanol when grown in a liquid medium at 37° C. Moreover, microbial strains that have a tolerance to 2-butanol and isobutanol are not known in the art. Therefore, identification of microorganisms that have a high tolerance to 1-butanol, 2-butanol, and isobutanol would represent an advance in the art.

In addition, 2-butanone and ethanol are valuable compounds that can be produced by fermentation using microorganisms. 2-Butanone, also referred to as methyl ethyl ketone (MEK), is a widely used solvent and is the most important commercially produced ketone, after acetone. It is used as a solvent for paints, resins, and adhesives, as well as a selective extractant and activator of oxidative reactions. 2-butanone can be made by omitting the last step of the 2-butanol biosynthetic pathway (Donaldson et al., copending and commonly owned U.S. Patent Application No. 60/796,816). Ethanol is in high demand as an alternative fuel. Genetically modified strains of E. coli have been used as biocatalysts for ethanol production (Underwood et al., (2002) Appl. Environ. Microbiol. 68:6263-6272). A genetically modified strain of Zymomonas mobilis that has improved production of ethanol is described in US 2003/0162271 A1. Identification of microorganisms with improved tolerance to 2-butanone and ethanol would enhance the production of these compounds.

There is a need, therefore, for microbial host strains that are more tolerant to butanols and may be used for the bioproduction of butanols to high titer. The present invention addresses this need through the discovery of butanol tolerant microorganisms and development of methods for their isolation. In addition the discovered microorganisms have increased tolerance to 2-butanone and ethanol.

SUMMARY OF THE INVENTION

The invention relates to butanol tolerant microorganisms, particularly members of the genus Enterococcus, and methods for the isolation of the same. Microbial consortia were enriched and selected for tolerance to butanol. Several species of Enterococcus were isolated that demonstrated tolerance to concentrations of butanol of at least 2.5% w/v 1-butanol when grown on a solid medium at 37° C.

Accordingly, in one embodiment the invention provides a method for the isolation of a butanol tolerant microorganism comprising:

-   -   a) providing a microbial sample comprising a microbial         consortium;     -   b) contacting the microbial consortium in a growth medium         comprising a fermentable carbon source until the members of the         microbial consortium are growing;     -   c) contacting the growing microbial consortium of step (b) with         butanol; and     -   d) isolating the viable members of step (c) wherein a butanol         tolerant microorganism is isolated. In a preferred embodiment         viable members of the microbial consortium are isolated by a         process comprising the steps of:         -   i) growing the viable members of the consortium that has             been challenged with butanol in a liquid medium in the             absence of butanol; whereby the viable members multiply;         -   ii) growing the cells of step (i) in the presence of             butanol; and         -   iii) collecting the cells of step (ii) that grow in the             presence of butanol wherein a butanol tolerant microorganism             is isolated.

In another embodiment the invention provides butanol tolerant microorganisms isolated by the processes of the invention where preferred butanol tolerant microorganisms belong to the genus Enterococcus.

In another embodiment the invention provides a method for the isolation of a butanol tolerant Enterococcus comprising:

-   -   a) providing a microbial sample comprising a microbial         consortium;     -   b) enriching the microbial consortium for the presence of         Enterococcus in a growth medium comprising:         -   i) a fermentable carbon source; and         -   ii) a competitor growth inhibitor;         -   to generate an Enterococcus enriched culture in which             members of the Enterococcus enriched culture are growing;     -   c) contacting the growing Enterococcus enriched culture of         step (b) with butanol; and     -   d) isolating the viable members of step (c) wherein a butanol         tolerant Enterococcus is isolated.

In another embodiment the invention provides a butanol tolerant Enterococcus having the following characteristics:

-   -   i) is a facultative anaerobe; and     -   ii) is Gram-positive; and     -   iii) is non-motile; and     -   iv) has spherical or ovoid cell morphology; and     -   v) has ability to initiate growth in 6.5% sodium chloride broth         and in broth at pH 9.6; and     -   vi) is tolerant to at least 2.5% w/v butanol when grown on a         solid medium.

In another embodiment the invention provides a butanol tolerant Enterococcus selected from the group consisting of ATCC PTA-7478 (Enterococcus faecium PN0110), ATCC_PTA-7479 (Enterococcus gallinarium PN0048), ATCC PTA-7477 (Enterococcus faecalis PN0197-10) and ATCC PTA-7480 (Enterococcus faecium PN0133).

In another embodiment the invention provides a method for the production of 2-butanone comprising:

-   -   a) providing an Enterococcus comprising genetic constructs         encoding a 2-butanone biosynthetic pathway, having the following         characteristics:         -   i) is a facultative anaerobe; and         -   ii) is Gram-positive; and         -   iii) is non-motile; and         -   iv) has spherical or ovoid cell morphology; and         -   v) has ability to initiate growth in 6.5% sodium chloride             broth and in broth at pH 9.6; and         -   vi) is tolerant to at least 2.5% w/v butanol when grown on a             solid medium and     -   b) growing the Enterococcus of step (a) under conditions whereby         2-butanone is produced.

BRIEF DESCRIPTION BIOLOGICAL DEPOSITS AND SEQUENCE DESCRIPTIONS

The various embodiments of the invention can be more fully understood from the following detailed description, biological deposits, and the accompanying sequence descriptions, which form a part of this application.

Applicants made the following biological deposits under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure:

International Depositor Identification Depository Reference Designation Date of Deposit Enterococcus faecium ATCC: PTA-7478 Apr. 7, 2006 PN0110 Enterococcus gallinarium ATCC: PTA-7479 Apr. 7, 2006 PN0048 Enterococcus faecalis ATCC: PTA-7477 Apr. 7, 2006 PN0197-10 Enterococcus faecium ATCC: PTA-7480 Apr. 7, 2006 PN0133

The following sequences conform with 37 C.F.R. 1.821-1.825 (“Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”) and are consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5 (a-bis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.

TABLE 1 Summary of Gene and Protein SEQ ID Numbers for 1-Butanol Biosynthetic Pathway SEQ ID NO: SEQ ID Nucleic NO: Description acid Peptide Acetyl-CoA acetyltransferase thlA 1 2 from Clostridium acetobutylicum ATCC 824 Acetyl-CoA acetyltransferase thlB 3 4 from Clostridium acetobutylicum ATCC 824 3-Hydroxybutyryl-CoA 5 6 dehydrogenase from Clostridium acetobutylicum ATCC 824 Crotonase from Clostridium 7 8 acetobutylicum ATCC 824 Putative trans-enoyl CoA 9 10 reductase from Clostridium acetobutylicum ATCC 824 Butyraldehyde dehydrogenase 11 12 from Clostridium beijerinckii NRRL B594 1-Butanol dehydrogenase bdhB 13 14 from Clostridium acetobutylicum ATCC 824 1-Butanol dehydrogenase 15 16 bdhA from Clostridium acetobutylicum ATCC 824

TABLE 2 Summary of Gene and Protein SEQ ID Numbers for 2-Butanol Biosynthetic Pathway SEQ ID NO: SEQ ID Nucleic NO: Description acid Peptide budA, acetolactate decarboxylase 17 18 from Klebsiella pneumoniae ATCC 25955 budB, acetolactate synthase from 19 20 Klebsiella pneumoniae ATCC 25955 budC, butanediol dehydrogenase 21 22 from Klebsiella pneumoniae IAM1063 pddA, butanediol dehydratase 23 24 alpha subunit from Klebsiella oxytoca ATCC 8724 pddB, butanediol dehydratase 25 26 beta subunit from Klebsiella oxytoca ATCC 8724 pddC, butanediol dehydratase 27 28 gamma subunit from Klebsiella oxytoca ATCC 8724 sadH, 2-butanol dehydrogenase 29 30 from Rhodococcus ruber 219

TABLE 3 Summary of Gene and Protein SEQ ID Numbers for Isobutanol Biosynthetic Pathway SEQ ID NO: SEQ ID Nucleic NO: Description acid Peptide Klebsiella pneumoniae budB 19 20 (acetolactate synthase) E. coli ilvC (acetohydroxy acid 31 32 reductoisomerase) E. coli ilvD (acetohydroxy acid 33 34 dehydratase) Lactococcus lactis kivD 35 36 (branched-chain α-keto acid decarboxylase), codon optimized E. coli yqhD (branched-chain 37 38 alcohol dehydrogenase)

SEQ ID NOs:39 and 40 are the nucleotide sequences of primers used to amplify the 16S rRNA genes of butanol tolerant strains, as described in Example 1.

SEQ ID NOs:41-44 are the nucleotide sequences of the 16S rRNA genes of butanol tolerant Enterococcus strains, isolated as described in Examples 1-4.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides microorganisms that demonstrate high tolerance to alcohols, particularly butanols, as well as to 2-butanone and ethanol. The microorganisms of the invention are able to grow in the presence of 2.5% w/v or greater 1-butanol, 2-butanol, isobutanol, 2-butanone, or ethanol on a solid medium. Additionally, the invention provides a method for the isolation of butanol tolerant microorganisms. These butanol tolerant microorganisms may be genetically engineered to comprise a butanol biosynthetic pathway or a 2-butanone biosynthetic pathway, and used for the bioproduction of 1-butanol, 2-butanol, isobutanol, or 2-butanone to high titer.

The following definitions and abbreviations are to be used for the interpretation of the claims and the specification.

The term “butanol” as used herein, refers to 1-butanol, 2-butanol, isobutanol, or mixtures thereof.

The terms “butanol tolerant microorganism” and “tolerant” when used to describe a microorganism of the invention, refers to a bacterium or yeast that exhibits growth in the presence of 2.5% w/v or greater 1-butanol, 2-butanol, or isobutanol when grown on a solid medium at 37° C., or in the presence 2.0% w/v or greater 1-butanol, 2-butanol, or isobutanol when grown in a liquid medium at 37° C.

The term “microbial consortium” refers to a heterogeneous group of microbes with different genotypes. By way of example, a microbial consortium may be an environmental sample such as a wastewater sludge or soil or compost or contaminated water samples; a chemically mutagenized microbial population of a pure bacterial strain; a microbial strain containing a multicopy plasmid library; of a population of transposon tagged mutants of a particular strain.

The term “environmental sample” refers to a sample obtained from the environment. In particular, the environmental sample may be wastewater sludge or other sample obtained from an environment where there has been exposure to butanol and/or other solvents. The environmental sample comprises a microbial consortium.

The term “enriching” as applied to a microbial culture and particularly the culturing of a microbial consortium refers to the practice of supplying the cells of the consortium or microbial culture with an excess of growth nutrients to enhance or encourage the growth of the cells.

The terms “fermentable carbon source”, “carbon substrate” or “fermentable carbon substrate” are used interchangeably and refer to a source of carbon that is readily utilized by a microbial consortium. Fermentable carbon sources include, but are not limited to, monosaccharides, oligosaccharides, polysaccharides, and one-carbon substrates or mixtures thereof. A non-limiting list of preferred fermentable carbon sources includes simple sugars, such as glucose, fructose, and sucrose; and carboxylic acids such as fatty acids, butyric acid, and valeric acid.

The term “aerobic conditions” means growth conditions in the presence of oxygen.

The term “anaerobic conditions” means growth conditions in the absence of oxygen.

The term “microaerophilic conditions” means growth conditions with low levels of oxygen (i.e., below normal atmospheric oxygen levels).

The term “butanol biosynthetic pathway” refers to an enzyme pathway to produce 1-butanol, 2-butanol, or isobutanol.

The term “1-butanol biosynthetic pathway” refers to an enzyme pathway to produce 1-butanol from acetyl-coenzyme A (acetyl-CoA).

The term “2-butanol biosynthetic pathway” refers to an enzyme pathway to produce 2-butanol from pyruvate.

The term “2-butanone biosynthetic pathway” refers to the enzyme pathways to produce 2-butanone from pyruvate.

The term “isobutanol biosynthetic pathway” refers to an enzyme pathway to produce isobutanol from pyruvate.

The term “acetyl-CoA acetyltransferase” refers to an enzyme that catalyzes the conversion of two molecules of acetyl-CoA to acetoacetyl-CoA and coenzyme A (CoA). Preferred acetyl-CoA acetyltransferases are acetyl-CoA acetyltransferases with substrate preferences (reaction in the forward direction) for a short chain acyl-CoA and acetyl-CoA and are classified as E.C. 2.3.1.9 [Enzyme Nomenclature 1992, Academic Press, San Diego]; although, enzymes with a broader substrate range (E.C. 2.3.1.16) will be functional as well. Acetyl-CoA acetyltransferases are available from a number of sources, for example, Escherichia coli (GenBank Nos: NP_(—)416728, NC_(—)000913; NCBI (National Center for Biotechnology Information) amino acid sequence, NCBI nucleotide sequence), Clostridium acetobutylicum (GenBank Nos: NP_(—)349476.1 (SEQ ID NO:2), NC_(—)003030; NP_(—)149242 (SEQ ID NO:4), NC_(—)001988), Bacillus subtilis (GenBank Nos: NP_(—)390297, NC_(—)000964), and Saccharomyces cerevisiae (GenBank Nos: NP_(—)015297, NC_(—)001148).

The term “3-hydroxybutyryl-CoA dehydrogenase” refers to an enzyme that catalyzes the conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA. 3-Hydroxybutyryl-CoA dehydrogenases may be reduced nicotinamide adenine dinucleotide (NADH)-dependent, with a substrate preference for (S)-3-hydroxybutyryl-CoA or (R)-3-hydroxybutyryl-CoA and are classified as E.C. 1.1.1.35 and E.C. 1.1.1.30, respectively. Additionally, 3-hydroxybutyryl-CoA dehydrogenases may be reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent, with a substrate preference for (S)-3-hydroxybutyryl-CoA or (R)-3-hydroxybutyryl-CoA and are classified as E.C. 1.1.1.157 and E.C. 1.1.1.36, respectively. 3-Hydroxybutyryl-CoA dehydrogenases are available from a number of sources, for example, C. acetobutylicum (GenBank NOs: NP_(—)349314 (SEQ ID NO:6), NC_(—)003030), B. subtilis (GenBank NOs: AAB09614, U29084), Ralstonia eutropha (GenBank NOs: ZP_(—)0017144, NZ_AADY01000001, Alcaligenes eutrophus (GenBank NOs: YP_(—)294481, NC_(—)007347), and A. eutrophus (GenBank NOs: P14697, J04987).

The term “crotonase” refers to an enzyme that catalyzes the conversion of 3-hydroxybutyryl-CoA to crotonyl-CoA and H₂O. Crotonases may have a substrate preference for (S)-3-hydroxybutyryl-CoA or (R)-3-hydroxybutyryl-CoA and are classified as E.C. 4.2.1.17 and E.C. 4.2.1.55, respectively. Crotonases are available from a number of sources, for example, E. coli (GenBank NOs: NP_(—)415911 (SEQ ID NO:8), NC_(—)000913), C. acetobutylicum (GenBank NOs: NP_(—)349318, NC_(—)003030), B. subtilis (GenBank NOs: CAB13705, Z99113), and Aeromonas caviae (GenBank NOs: BAA21816, D88825).

The term “butyryl-CoA dehydrogenase”, also called trans-enoyl CoA reductase, refers to an enzyme that catalyzes the conversion of crotonyl-CoA to butyryl-CoA. Butyryl-CoA dehydrogenases may be NADH-dependent or NADPH-dependent and are classified as E.C. 1.3.1.44 and E.C. 1.3.1.38, respectively. Butyryl-CoA dehydrogenases are available from a number of sources, for example, C. acetobutylicum (GenBank NOs: NP_(—)347102 (SEQ ID NO:10), NC_(—)003030), Euglena gracilis (GenBank NOs: Q5EU90, AY741582), Streptomyces collinus (GenBank NOs: AAA92890, U37135), and Streptomyces coelicolor (GenBank NOs: CAA22721, AL939127).

The term “butyraldehyde dehydrogenase” refers to an enzyme that catalyzes the conversion of butyryl-CoA to butyraldehyde, using NADH or NADPH as cofactor. Butyraldehyde dehydrogenases with a preference for NADH are known as E.C. 1.2.1.57 and are available from, for example, Clostridium beijerinckii (GenBank NOs: AAD31841 (SEQ ID NO:12), AF157306) and C. acetobutylicum (GenBank NOs: NP_(—)149325, NC_(—)001988).

The term “1-butanol dehydrogenase” refers to an enzyme that catalyzes the conversion of butyraldehyde to 1-butanol. 1-butanol dehydrogenases are a subset of the broad family of alcohol dehydrogenases. 1-butanol dehydrogenase may be NADH- or NADPH-dependent. 1-butanol dehydrogenases are available from, for example, C. acetobutylicum (GenBank NOs: NP_(—)149325, NC_(—)001988; NP_(—)349891 (SEQ ID NO:14), NC_(—)003030; and NP_(—)349892 (SEQ ID NO:16), NC_(—)003030) and E. coli (GenBank NOs: NP_(—)417-484, NC_(—)000913).

The term “acetolactate synthase”, also known as “acetohydroxy acid synthase”, refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of two molecules of pyruvic acid to one molecule of alpha-acetolactate. Acetolactate synthase, known as EC 2.2.1.6 [formerly 4.1.3.18] (Enzyme Nomenclature 1992, Academic Press, San Diego) may be dependent on the cofactor thiamin pyrophosphate. Suitable acetolactate synthase enzymes are available from a number of sources, for example, Bacillus subtilis (GenBank Nos: AAA22222 NCBI (National Center for Biotechnology Information) amino acid sequence, L04470 NCBI nucleotide sequence), Klebsiella terrigena (GenBank Nos: AAA25055, L04507), and Klebsiella pneumoniae (GenBank Nos: AAA25079 (SEQ ID NO:20), M73842 (SEQ ID NO:19).

The term “acetolactate decarboxylase” refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of alpha-acetolactate to acetoin. Acetolactate decarboxylases are known as EC 4.1.1.5 and are available, for example, from Bacillus subti/is (GenBank Nos: AAA22223, L04470), Klebsiella terrigena (GenBank Nos: AAA25054, L04507) and Klebsiella pneumoniae (SEQ ID NO:18 (amino acid) SEQ ID NO:17 (nucleotide)).

The term “butanediol dehydrogenase” also known as “acetoin reductase” refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of acetoin to 2,3-butanediol. Butanediol dehydrogenases are a subset of the broad family of alcohol dehydrogenases. Butanediol dehydrogenase enzymes may have specificity for production of R- or S-stereochemistry in the alcohol product. S-specific butanediol dehydrogenases are known as EC 1.1.1.76 and are available, for example, from Klebsiella pneumoniae (GenBank Nos: BBA13085 (SEQ ID NO:22), D86412. R-specific butanediol dehydrogenases are known as EC 1.1.1.4 and are available, for example, from Bacillus cereus (GenBank Nos. NP_(—)830481, NC_(—)004722; AAP07682, AE017000), and Lactococcus lactis (GenBank Nos. AAK04995, AE006323).

The term “butanediol dehydratase”, also known as “diol dehydratase” or “propanediol dehydratase” refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of 2,3-butanediol to 2-butanone, also known as methyl ethyl ketone (MEK). Butanediol dehydratase may utilize the cofactor adenosyl cobalamin. Adenosyl cobalamin-dependent enzymes are known as EC 4.2.1.28 and are available, for example, from Klebsiella oxytoca (GenBank Nos: BAA08099 (alpha subunit) (SEQ ID NO:24), BAA08100 (beta subunit) (SEQ ID NO:26), and BBA08101 (gamma subunit) (SEQ ID NO:28), (Note all three subunits are required for activity), D45071).

The term “2-butanol dehydrogenase” refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of 2-butanone to 2-butanol. 2-butanol dehydrogenases are a subset of the broad family of alcohol dehydrogenases. 2-butanol dehydrogenase may be NADH- or NADPH-dependent. The NADH-dependent enzymes are known as EC 1.1.1.1 and are available, for example, from Rhodococcus ruber (GenBank Nos: CAD36475 (SEQ ID NO:30), AJ491307 (SEQ ID NO:29)). The NADPH-dependent enzymes are known as EC 1.1.1.2 and are available, for example, from Pyrococcus furiosus (GenBank Nos: AAC25556, AF013169).

The term “acetohydroxy acid isomeroreductase” or “acetohydroxy acid reductoisomerase” refers to an enzyme that catalyzes the conversion of acetolactate to 2,3-dihydroxyisovalerate using NADPH (reduced nicotinamide adenine dinucleotide phosphate) as an electron donor. Preferred acetohydroxy acid isomeroreductases are known by the EC number 1.1.1.86 and sequences are available from a vast array of microorganisms, including, but not limited to, Escherichia coli (GenBank Nos: NP_(—)418222 (SEQ ID NO:32), NC_(—)000913 (SEQ ID NO:31)), Saccharomyces cerevisiae (GenBank Nos: NP_(—)013459, NC_(—)001144), Methanococcus maripaludis (GenBank Nos: CAF30210, BX957220), and Bacillus subtilis (GenBank Nos: CAB14789, Z99118).

The term “acetohydroxy acid dehydratase” refers to an enzyme that catalyzes the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate. Preferred acetohydroxy acid dehydratases are known by the EC number 4.2.1.9. These enzymes are available from a vast array of microorganisms, including, but not limited to, E. coli (GenBank Nos: YP_(—)026248 (SEQ ID NO:34), NC_(—)000913 (SEQ ID NO:33)), S. cerevisiae (GenBank Nos: NP_(—)012550, NC_(—)001142), M. maripaludis (GenBank Nos: CAF29874, BX957219), and B. subtilis (GenBank Nos: CAB14105, Z99115).

The term “branched-chain α-keto acid decarboxylase” refers to an enzyme that catalyzes the conversion of α-ketoisovalerate to isobutyraldehyde and CO₂. Preferred branched-chain α-keto acid decarboxylases are known by the EC number 4.1.1.72 and are available from a number of sources, including, but not limited to, Lactococcus lactis (GenBank Nos: AAS49166, AY548760; CAG34226 (SEQ ID NO:36), AJ746364, Salmonella typhimurium (GenBank Nos: NP_(—)461346, NC_(—)003197), and Clostridium acetobutylicum (GenBank Nos: NP_(—)149189, NC_(—)001988).

The term “branched-chain alcohol dehydrogenase” refers to an enzyme that catalyzes the conversion of isobutyraldehyde to isobutanol. Preferred branched-chain alcohol dehydrogenases are known by the EC number 1.1.1.265, but may also be classified under other alcohol dehydrogenases (specifically, EC 1.1.1.1 or 1.1.1.2). These enzymes utilize NADH (reduced nicotinamide adenine dinucleotide) and/or NADPH as electron donor and are available from a number of sources, including, but not limited to, S. cerevisiae (GenBank Nos: NP_(—)010656, NC_(—)001136; NP_(—)014051, NC_(—)001145), E. coli (GenBank Nos: NP_(—)417484 (SEQ ID NO:38), NC_(—)000913 (SEQ ID NO:37)), and C. acetobutylicum (GenBank Nos: NP_(—)349892, NC_(—)003030).

The term “gene” refers to a nucleic acid fragment that is capable of being expressed as a specific protein, optionally including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure.

As used herein the term “coding sequence” refers to a DNA sequence that codes for a specific amino acid sequence. “Suitable regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing site, effector binding site and stem-loop structure.

The term “promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.

The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of effecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

The term “expression”, as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide.

As used herein the term “transformation” refers to the transfer of a nucleic acid fragment into a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “recombinant” or “transformed” organisms.

The terms “plasmid” and “vector” refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA fragments. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell. “Transformation vector” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitates transformation of a particular host cell.

As used herein the term “codon degeneracy” refers to the nature in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.

The term “codon-optimized” as it refers to genes or coding regions of nucleic acid molecules for transformation of various hosts, refers to the alteration of codons in the gene or coding regions of the nucleic acid molecules to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the DNA.

Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Man iatis, T., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory Cold Press Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987).

The term “invention” or “present invention” as used herein is meant to apply generally to all embodiments of the invention as described in the claims as presented or as later amended and supplemented, or in the specification.

In one embodiment the present invention provides a method for the isolation of butanol tolerant microorganisms. The method comprises enriching a microbial consortium under growth conditions and contacting the enriched consortium with butanol, as described in detail below. Microorganisms that demonstrate high tolerance to alcohols, particularly butanols, identified by the method of the invention, are also provided. These butanol tolerant microorganisms may be genetically engineered to comprise a butanol biosynthetic pathway and used for the bioproduction of 1-butanol, 2-butanol, or isobutanol to high titer.

Isolation of Butanol Tolerant Microorganisms

Butanol tolerant microorganisms may be isolated from environmental samples such as wastewater sludge and samples from other environments where there is exposure to butanol and/or other solvents. For example, environmental samples may be obtained from wastewater treatment facilities at chemical plants. Industrial wastewater bioreactors are particularly good sources of environmental samples of microorganisms with desirable resistance phenotypes because of the long-term growth in the presence of various organic solvents (Bramucci et al., Trends Biotechnol. 18:501-505 (2000)). Butanol tolerant microorganisms may be isolated from other microbial samples as well. For example, the microbial sample may be a chemically mutagenized microbial population of a pure bacterial strain, a microbial strain containing a multicopy plasmid library, or a transposon tagged mutants of a particular strain. Any of these microbial samples including a mixed population is said to include a microbial consortium.

In one embodiment of the present invention, the microbial sample is cultured in a growth medium to enrich the microbial consortium contained therein until the members of the consortium are growing. In one embodiment the cultures are growing in log phase. The growth medium comprises a fermentable carbon source and may include suitable levels of nitrogen, phosphorus, sulfur, and salts. Suitable levels of these nutrients necessary for growth of the microbial consortium are well known to those skilled in the art, and non-limiting examples are provided below. The fermentable carbon source may be any carbon source that is readily metabolized by the members of the microbial consortium, including but not limited to, sucrose, fructose, glucose, and mixtures thereof. The fermentable carbon source may also be a carboxylic acid such as a fatty acid, butyric acid or valeric acid. Typically, the carbon source is present at a concentration from about 0.1% weight/volume w/v to about 1.5% w/v. The nitrogen source may be any suitable nitrogen source, including but not limited to, ammonium salts or yeast extract. The nitrogen source is typically present in the growth medium at a concentration of about 10 mM. Phosphorus may be present in the medium in the form of phosphate salts, such as sodium and potassium phosphates, which are typically present in the growth medium at a concentration of about 50 mM. Sulfur may be present in the medium in the form of sulfate salts, such as sodium or ammonium sulfates, which are typically present in the growth medium at a concentration of about 10 mM. Additional salts include, but are not limited to, magnesium chloride, calcium chloride, manganese chloride, ferric chloride, ferrous chloride, zinc chloride, cupric chloride, cobalt chloride, and sodium molybdate. These salts are typically present in the growth medium at a concentration of about 1 mM to about 2 mM. The growth medium may also contain vitamins such as thiamine hydrochloride.

The enrichment culture is grown at a temperature of about 25° C. to about 60° C. for a time sufficient for the members of the microbial consortium in the sample to exhibit growth, typically about 12 hours to about 24 hours. The culture may be grown under anaerobic, microaerophilic, or aerobic conditions, with or without agitation. As is readily understood by the skilled person, anaerobic conditions are those that are devoid of oxygen, aerobic conditions are those that contain oxygen and microaerophilic conditions are those where oxygen is present at a level below that found in air, ie. less than 21%. Growth of the culture may be monitored by measuring the optical density, typically at a wavelength of 600 nm.

The growing enrichment culture is then contacted with butanol. This contacting may be done by diluting the enrichment culture with a fresh growth medium that contains butanol. It is particularly suitable if the enrichment culture is growing in log phase at this point. The butanol concentration used is about 0.8% w/v to about 3.0% w/v, preferably about 0.8% w/v to about 2.0% w/v. In one embodiment, the butanol is predominantly 1-butanol. In another embodiment, the butanol is predominantly 2-butanol. In another embodiment, the butanol is predominantly isobutanol. As used herein, predominantly means at least about 90% by weight of the total butanol. Additionally, mixtures comprising various combinations of two or more of 1-butanol, 2-butanol, and isobutanol may be used. The culture is grown for a period of time until significant growth is observed. Optionally, the cultures that demonstrate significant growth may be contacted with butanol again one or more times to select for increased tolerance to butanol. Each contacting may be made with progressively higher butanol concentrations.

The microbial consortium that was contacted with butanol is then separated to isolate individual strains. Multiple means of cell isolation are know to those skilled in the art involving either liquid or solid media. For example, the microbial consortium that was contacted with butanol may be plated onto a solid medium, for example nutrient agar, Luria Bertani (LB) agar, modified LB agar (i.e., LB agar supplemented with a fermentable carbon source and salts), or minimal enrichment medium with agar, which may or may not contain butanol. If butanol is present in the solid medium, its concentration is typically about 1.2% w/v to about 3% w/v. The culture is grown until colonies are formed. The colonies are then isolated using methods known in the art to provide a butanol tolerant microorganism. For example, the colonies from the solid medium may be collected and identified using methods known in the art, as described below. Alternatively, the colonies from the solid medium may be inoculated into a growth medium (e.g., minimal enrichment medium), either liquid or solid, that does not contain butanol. After growth, the cells may be collected and identified. Optionally, the cells from the colonies may be grown in the presence of butanol, either in liquid or solid growth medium (e.g., minimal enrichment medium). Typically, the butanol concentration in the medium is about 1.2% w/v to about 3% w/v. The cells that grow in the presence of butanol are collected. The isolated microorganisms may be identified using methods known in the art, such as 16S ribosomal RNA (rRNA) gene sequencing, fatty acid profile analysis, or ribotyping.

The butanol tolerant microorganisms isolated by the method of the present invention are tolerant to at least 2.5% butanol (i.e., 1-butanol, 2-butanol, or isobutanol) when grown on a solid medium at 37° C., or to at least 2.0% w/v butanol when grown in a liquid medium at 37° C. It should be noted that the butanol tolerance of microorganisms is typically higher when grown on a solid medium than when grown on a liquid medium. Additionally, the butanol tolerance of microorganisms is dependent on the growth temperature, typically being higher at lower growth temperatures. Microorganisms isolated by contacting the enriched microbial consortium with one butanol are generally also tolerant to other butanols. For example, microorganisms isolated using 1-butanol are also tolerant to 2-butanol and isobutanol.

The enrichment culture may also be grown and contacted with butanol in a continuous culture in a chemostat bioreactor. The cells in a chemostat bioreactor can be grown at various growth rates by appropriate adjustment of the dilution rate. Chemostat cultures can be precisely controlled for aeration and pH, leading to higher cell densities. Additionally, butanol can be gradually added in increasing concentration by adjusting feed composition. After contacting the enrichment culture with butanol in the bioreactor, the butanol tolerant microorganisms are isolated and identified as described above.

Unexpectedly, the majority of the butanol tolerant microorganisms identified using the method of the present invention in the examples herein were bacteria belonging to the genus Enterococcus. Enterococcus are facultively anaerobic, Gram-positive, non-motile, spherical or ovoid cells having the ability to initiate growth in 6.5% sodium chloride broth and in broth at pH 9.6 (Bergey's Manual of Systematic Bacteriology, Vol 2, Sneath et al., Eds.; Williams & Wilkins, Baltimore, Md., 1986, pp. 1063-1065). The butanol tolerant Enterococcus strains of the present invention are further characterized by the 16S rRNA gene sequences given as SEQ ID NOs:41, 42, 43, and 44.

The present method for isolation of butanol tolerant microorganisms may be modified to selectively isolate butanol tolerant Enterococcus. In this embodiment, the microbial consortium from the environmental sample is enriched for the presence of Enterococcus in a growth medium comprising a fermentable carbon source, suitable levels of nitrogen, phosphorus, sulfur, and salts, as described above, and a “competitor growth inhibitor” which functions to inhibit the growth of microorganisms other than Enterococcus. Suitable competitor growth inhibitors include, but are not limited to, bile, azide, and mixtures thereof. The optimum amount of the competitor growth inhibitor to use will depend on the microbial consortium and the type of inhibitor, and can be determined using routine experimentation. The Enterococcus enriched culture is grown to log phase and contacted with butanol as described above. The butanol tolerant Enterococcus strains are then isolated and identified as described above.

The isolated butanol tolerant Enterococcus strains may be genetically engineered to comprise genetic constructs encoding a butanol biosynthetic pathway and grown under suitable conditions to produce butanol. The butanol biosynthetic pathway may be a 1-butanol, 2-butanol, or isobutanol biosynthetic pathway.

1-Butanol Biosynthetic Pathway

A biosynthetic pathway for the production of 1-butanol is described by Donaldson et al. in copending and commonly owned U.S. patent application Ser. No. 11/527,995, which is incorporated herein by reference. This biosynthetic pathway comprises the following substrate to product conversions:

-   -   a) acetyl-CoA to acetoacetyl-CoA, as catalyzed for example by         acetyl-CoA acetyltransferase encoded by the genes given as SEQ         ID NO:1 or 3;     -   b) acetoacetyl-CoA to 3-hydroxybutyryl-CoA, as catalyzed for         example by 3-hydroxybutyryl-CoA dehydrogenase encoded by the         gene given as SEQ ID NO:5;     -   c) 3-hydroxybutyryl-CoA to crotonyl-CoA, as catalyzed for         example by crotonase encoded by the gene given as SEQ ID NO:7;     -   d) crotonyl-CoA to butyryl-CoA, as catalyzed for example by         butyryl-CoA dehydrogenase encoded by the gene given as SEQ ID         NO:9;     -   e) butyryl-CoA to butyraldehyde, as catalyzed for example by         butyraldehyde dehydrogenase encoded by the gene given as SEQ ID         NO:11; and     -   f) butyraldehyde to 1-butanol, as catalyzed for example by         1-butanol dehydrogenase encoded by the genes given as SEQ ID         NO:13 or 15.

The pathway requires no ATP and generates NAD⁺ and/or NADP⁺, thus, it balances with the central metabolic routes that generate acetyl-CoA.

2-Butanol and 2-Butanone Biosynthetic Pathway Biosynthetic pathways for the production of 2-butanol and 2-butanone are described by Donaldson et al. in copending and commonly owned U.S. Patent Application No. 60/796,816, which is incorporated herein by reference. One 2-butanol biosynthetic pathway comprises the following substrate to product conversions:

-   -   a) pyruvate to alpha-acetolactate, as catalyzed for example by         acetolactate synthase encoded by the gene given as SEQ ID NO:19;     -   b) alpha-acetolactate to acetoin, as catalyzed for example by         acetolactate decarboxylase encoded by the gene given as SEQ ID         NO:17;     -   c) acetoin to 2,3-butanediol, as catalyzed for example by         butanediol dehydrogenase encoded by the gene given as SEQ ID         NO:21;     -   d) 2,3-butanediol to 2-butanone, catalyzed for example by         butanediol dehydratase encoded by genes given as SEQ ID NOs:23,         25, and 27; and     -   e) 2-butanone to 2-butanol, as catalyzed for example by         2-butanol dehydrogenase encoded by the gene given as SEQ ID         NO:29.     -   Omitting the last step (e) of the above pathway provides a         biosynthetic pathway for production of 2-butanone, also known as         methyl ethyl ketone (MEK),         Isobutanol Biosynthetic Pathway

Biosynthetic pathways for the production of isobutanol are described by Maggio-Hall et al. in copending and commonly owned U.S. patent application Ser. No. 11/586,315, which is incorporated herein by reference. One isobutanol biosynthetic pathway comprises the following substrate to product conversions:

-   -   a) pyruvate to acetolactate, as catalyzed for example by         acetolactate synthase encoded by the gene given as SEQ ID NO:19;     -   b) acetolactate to 2,3-dihydroxyisovalerate, as catalyzed for         example by acetohydroxy acid isomeroreductase encoded by the         gene given as SEQ ID NO:31;     -   c) 2,3-dihydroxyisovalerate to α-ketoisovalerate, as catalyzed         for example by acetohydroxy acid dehydratase encoded by the gene         given as SEQ ID NO:33;     -   d) α-ketoisovalerate to isobutyraldehyde, as catalyzed for         example by a branched-chain keto acid decarboxylase encoded by         the gene given as SEQ ID NO:35; and     -   e) isobutyraldehyde to isobutanol, as catalyzed for example by a         branched-chain alcohol dehydrogenase encoded by the gene given         as SEQ ID NO:37.         Construction of Enterococcus Hosts for Butanol Production

Recombinant, butanol tolerant Enterococcus strains containing the necessary genes that will encode one of the enzymatic pathways for the conversion of a fermentable carbon substrate to butanol may be constructed using techniques well known in the art. The genome sequences of Enterococcus faecalis and Enterococcus faecium are known (National Center for Biotechnology Information (NCBI) database). These bacteria have a G+C content of 37%. Enterococcus gallinarum is closely related to Enterococcus faecalis and Enterococcus faecium.

In the present invention, genes encoding the enzymes of one of the butanol biosynthetic pathways described above may be isolated from various sources (see above). Methods of obtaining desired genes from a bacterial genome are common and well known in the art of molecular biology. For example, if the sequence of the gene is known, primers may be designed and the desired sequence amplified using standard primer-directed amplification methods such as polymerase chain reaction (U.S. Pat. No. 4,683,202) to obtain amounts of DNA suitable for cloning into transformation vectors. If a gene that is heterologous to a known sequence is to be isolated, suitable genomic libraries may be created by restriction endonuclease digestion and may be screened with probes having complementary sequence to the desired gene sequence. Once the sequence is isolated, the DNA may be amplified using standard primer-directed amplification methods such as polymerase chain reaction (U.S. Pat. No. 4,683,202) to obtain amounts of DNA suitable for transformation using appropriate vectors. Tools for codon optimization for expression in a heterologous host are readily available.

Once the relevant pathway genes are identified and isolated they may be inserted in a vector and transformed into a butanol tolerant Enterococcus host by means well known in the art. Vectors useful for the transformation of Enterococcus are known (see below). Typically the vector contains sequences directing transcription and translation of the inserted DNA fragment, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5′ of the inserted DNA fragment which harbors transcriptional initiation controls and a region 3′ of the DNA fragment which controls transcriptional termination. Both control regions may be derived from genes homologous to the transformed host cell, although it is to be understood that such control regions may also be derived from genes that are not native to the specific production host.

Initiation control regions or promoters, which are useful to drive expression of the relevant pathway coding regions in the desired Enterococcus host cell, may be obtained from other lactic acid bacteria or other Gram-positive organisms. A non-limiting example is the nisA promoter from Lactococcus. Termination control regions may also be derived from various genes native to the preferred hosts or related bacteria. Optionally, a termination site may be unnecessary, however, it is most preferred if included.

The Enterococcus genus belongs to the Lactobacillales family and many plasmids and vectors used in the transformation of Lactobacillus, Bacillus subtilis, and Streptococcus may be used for Enterococcus. Non-limiting examples of suitable vectors include pAMβ1 and derivatives thereof (Renault et al., Gene 183:175-182 (1996); and O'Sullivan et al., Gene 137:227-231 (1993)); pMBB1 and pHW800, a derivative of pMBB1 (Wyckoff et al. Appl. Environ. Microbiol. 62:1481-1486 (1996)); pMG1, a conjugative plasmid (Tanimoto et al., J. Bacteriol. 184:5800-5804 (2002)); pNZ9520 (Kleerebezem et al., Appl. Environ. Microbiol. 63:4581-4584 (1997)); pAM401 (Fujimoto et al., Appl. Environ. Microbiol. 67:1262-1267 (2001)); and pAT392 (Arthur et al., Antimicrob. Agents Chemother. 38:1899-1903 (1994)). Expression vectors for E. faecalis using the nisA promoter from Lactococcus may also be used (Eichenbaum et al., Appl. Environ. Microbiol. 64:2763-2769 (1998). Additionally, vectors for gene replacement in the E. faecium chromosome may be used (Nallaapareddy et al., Appl. Environ. Microbiol. 72:334-345 (2006)).

The various genes for a butanol biosynthetic pathway may be assembled into any suitable vector, such as those described above. The codons can be optimized for expression based on the codon index deduced from the genome sequences of Enterococcus faecalis or Enterococcus faecium. The plasmids may be introduced into the butanol tolerant host cell using methods known in the art, such as electroporation, as described by Cruz-Rodz et al. (Molecular Genetics and Genomics 224:1252-154 (1990)) or conjugation, as described by Tanimoto et al. (J. Bacteriol. 184:5800-5804 (2002)) and Grohamann et al. (Microbiol. Mol. Biol. Rev. 67:277-301 (2003)).

Fermentation Media

Fermentation media for the production of butanol must contain suitable carbon substrates. Suitable substrates may include but are not limited to monosaccharides such as glucose and fructose, oligosaccharides such as lactose or sucrose, polysaccharides such as starch or cellulose or mixtures thereof and unpurified mixtures from renewable feedstocks such as cheese whey permeate, cornsteep liquor, sugar beet molasses, and barley malt.

Although it is contemplated that all of the above mentioned carbon substrates and mixtures thereof are suitable in the present invention, preferred carbon substrates are glucose, fructose, and sucrose.

In addition to an appropriate carbon source, fermentation media must contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the enzymatic pathway necessary for butanol production.

Culture Conditions

Typically cells are grown at a temperature in the range of about 25° C. to about 40° C. in an appropriate medium. Suitable growth media in the present invention are common commercially prepared media such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth or Yeast Medium (YM) broth. Other defined or synthetic growth media may also be used, and the appropriate medium for growth of the particular microorganism will be known by one skilled in the art of microbiology or fermentation science. The use of agents known to modulate catabolite repression directly or indirectly, e.g., cyclic adenosine 2′:3′-monophosphate, may also be incorporated into the fermentation medium.

Suitable pH ranges for the fermentation are between pH 5.0 to pH 9.0, where pH 6.0 to pH 8.0 is preferred as the initial condition.

Fermentations may be performed under aerobic or anaerobic conditions, where anaerobic or microaerobic conditions are preferred.

Industrial Batch and Continuous Fermentations

Butanol may be produced using a batch method of fermentation. Aclassical batch fermentation is a closed system where the composition of the medium is set at the beginning of the fermentation and not subject to artificial alterations during the fermentation. A variation on the standard batch system is the fed-batch system. Fed-batch fermentation processes are also suitable in the present invention and comprise a typical batch system with the exception that the substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the media. Batch and fed-batch fermentations are common and well known in the art and examples may be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass., or Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36:227, (1992), herein incorporated by reference.

Butanol may also be produced using continuous fermentation methods. Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth. Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. Methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology and a variety of methods are detailed by Brock, supra.

It is contemplated that the production of butanol may be practiced using either batch, fed-batch or continuous processes and that any known mode of fermentation would be suitable. Additionally, it is contemplated that cells may be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for butanol production.

Methods for Butanol Isolation from the Fermentation Medium

The bioproduced butanol may be isolated from the fermentation medium using methods known in the art for ABE fermentations (see for example, Durre, Appl. Microbiol. Biotechnol. 49:639-648 (1998), Groot et al., Process. Biochem. 27:61-75 (1992), and references therein). For example, solids may be removed from the fermentation medium by centrifugation, filtration, decantation, or the like. Then, the butanol may be isolated from the fermentation medium using methods such as distillation, azeotropic distillation, liquid-liquid extraction, adsorption, gas stripping, membrane evaporation, or pervaporation.

EXAMPLES

The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions.

The meaning of abbreviations used is as follows: “min” means minute(s), “h” means hour(s), “sec’ means second(s), “μL” means microliter(s), “mL” means milliliter(s), “L” means liter(s), “nm” means nanometer(s), “mm” means millimeter(s), “cm” means centimeter(s), “μm” means micrometer(s), “mM” means millimolar, “M” means molar, “mmol” means millimole(s), “μmole” means micromole(s), “g” means gram(s), “μg” means microgram(s), “mg” means milligram(s), “rpm” means revolutions per minute, “w/v” means weight/volume, “OD” means optical density, “OD₆₀₀” means optical density measured at a wavelength of 600 nm, “OD₅₉₅” means optical density measured at a wavelength of 595 nm, “IC₅₀” means the concentration of butanol that causes a 50% inhibition of growth, “GCMS” means gas chromatography-mass spectrometry, and “HPLC” means high performance liquid chromatography.

General Methods:

Standard recombinant DNA and molecular cloning techniques used in the Examples are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, by T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1984, and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience, N.Y., 1987.

Materials and methods suitable for the maintenance and growth of bacterial cultures are also well known in the art. Techniques suitable for use in the following Examples may be found in Manual of Methods for General Bacteriology, Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, eds., American Society for Microbiology, Washington, D.C., 1994, or by Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, Mass., 1989. All reagents, restriction enzymes and materials used for the growth and maintenance of bacterial cells were obtained from Aldrich Chemicals (Milwaukee, Wis.), BD Diagnostic Systems (Sparks, Md.), Life Technologies (Rockville, Md.), or Sigma Chemical Company (St. Louis, Mo.), unless otherwise specified.

Example 1 Isolation of Butanol Tolerant Microorganisms

The purpose of this Example was to isolate butanol tolerant microorganisms. Environmental samples were obtained from several wastewater treatment sites and were grown in enrichment cultures containing 1-butanol in shake flasks. A butanol tolerant bacterial strain was isolated and identified as Enterococcus faecium.

Environmental sludge samples were obtained from wastewater treatment facilities at several E.I. du Pont de Nemours and Company sites. Enrichment cultures were established for each sample by inoculating a 125-mL screw cap Erlenmeyer flask with 1 mL of a sludge sample in 10 mL of minimal enrichment medium (i.e., 10 mM ammonium sulfate, 50 mM potassium phosphate buffer, pH 7.0, 2 mM MgCl₂, 0.7 mM CaCl₂, 50 μM MnCl₂, 1 μM FeCl₃, 1 μM ZnCl₂, 1.72 μM CuCl₂, 2.53 μM CoCl₂, 2.42 μM Na₂MoO₄, 2 μM thiamine hydrochloride, 0.5% glucose, 0.5% fructose, 0.5% sucrose and 0.1% yeast extract). The enrichment cultures were incubated at 37° C. with shaking. After 24 h of incubation, the enrichment cultures were diluted 1:10 into fresh medium that contained 0.8%, 1.2% or 1.6% w/v 1-butanol (Sigma Chemical Co.; molecular biology grade (greater than or equal to 99% purity)). Incubation continued under the same conditions. Cultures with the highest level of 1-butanol allowing significant growth (e.g., a culture with an initial OD₆₀₀ of approximately 0.1 that increased within 24 to 48 h to an OD₆₀₀ of approximately 1.0) were then diluted 1:10 into fresh medium that contained higher levels of 1-butanol to select for increased tolerance to 1-butanol. Most of the enrichment cultures grew in the presence of 1.2% 1-butanol and enrichment cultures from some sites grew in the presence of 1.6% 1-butanol. However, none of the enrichment cultures grew in the presence of 2.0% 1-butanol in a liquid medium.

Pure strains of 1-butanol resistant bacteria were isolated from the enrichment cultures as follows. The cells grown in an enrichment culture containing 1.2% or 1.6% 1-butanol were serially diluted, and the serial dilutions were plated on Luria Bertani (LB) agar medium, modified LB agar (i.e., LB agar supplemented with 0.5% sucrose, 0.5% glucose, 0.5% fructose, 0.5% sodium pyruvate, 2 mM MgCl₂, 0.7 mM CaCl₂, 50 μM MnCl₂, 1 μM FeCl₃, 1 μM ZnCl₂, 1.72 μM CuCl₂, 2.53 μM CoCl₂, 2.42 μM Na₂MoO₄ and 2 μM thiamine hydrochloride) or minimal enrichment medium with 2.0% Difco™ Agar Noble, (BD Diagnostic Systems, Sparks, Md.), all without 1-butanol. Colonies were then inoculated from the agar medium into minimal enrichment medium (described above) without 1-butanol in the wells of a microtiter plate. The cells in each well were subcultured in separate microtiter plates with and without 1-butanol to identify 1-butanol-tolerant isolates. The amount of 1-butanol used to challenge the isolates ranged from 1.2% to 3.0% w/v. Several isolates from the enrichments displayed growth in 2.0% or higher 1-butanol in a liquid medium at 37° C. The IC₅₀ values of the isolated strains able to grow in liquid medium containing at least 2.0% 1-butanol were determined as follows. The growth was monitored (by OD₆₀₀) of isolates cultured in S30L medium (i.e., 10 mM ammonium sulfate, 5 mM potassium phosphate buffer, pH 7.0, 50 mM MOPS, pH 7.0, 2 mM MgCl₂, 0.7 mM CaCl₂, 50 μM MnCl₂, 1 μM FeCl₃, 1 μM ZnCl₂, 1.72 μM CuCl₂, 2.53 μM CoCl₂, 2.42 μM Na₂MoO₄, 2 μM thiamine hydrochloride, 0.01 M glucose, and 0.2% yeast extract) at 37° C. in the absence (control) and in the presence of from 1.2% to 3.0% w/v of 1-butanol and the doubling time for each culture was calculated from the logarithmic part of the growth curve (doubling time=0.693/growth rate). The percent growth inhibition caused by 1-butanol in the sample flasks was determined by subtracting the percent growth ([doubling time of the control flask/doubling time of the sample flask]×100) from 100%. The IC₅₀ was the concentration of butanol that caused 50% growth inhibition and was determined by plotting the concentration of butanol versus percent inhibition. The results for a representative strain that was able to grow on solid medium containing 2.5% w/v 1-butanol at 37° C. are summarized in Table 4. Higher IC₅₀ values were obtained when the strains were cultured at 30° C. (data not shown). The isolates were identified by sequencing the product that resulted from polymerase chain reaction (PCR) amplification of the 16S rRNA genes in DNA that was extracted from each isolate. DNA was extracted from each of the 1-butanol tolerant strains. Each isolate was processed using a commercial kit (Ultraclean Microbial Genomic DNA Isolation Kit, obtained from Mo Bio Laboratories, Inc, Carlsbad, Calif., Part No. 12224-50). The 16S rRNA genes of the isolates were amplified by PCR using HotStar Taq (Qiagen, Valencia, Calif.; Catalog No. 203446) with primers JCR14 (ACGGGCGGTGTGTAC), given as SEQ ID NO:39 and JCR15 (GCCAGCAGCCGCGGTA), given as SEQ ID NO:40. The PCR conditions were 15 min at 95° C., followed by 30 cycles at 94° C. for 45 sec, 55° C. for 1 min, and 72° C. for 1 min, followed by 10 min at 72° C. The PCR products were purified and sequenced. Each sequence was used as the query sequence for a BLAST search of GenBank for the most similar previously identified 16S rRNA gene sequence. The sequence of the rRNA gene of the isolated strain that was designated PN0110 (SEQ ID NO: 41) matched the known rRNA sequence for Enterococcus faecium (see Table 4).

TABLE 4 Butanol Tolerant Bacterial Strain Isolated from Environmental Samples IC₅₀ (%) ATTC 16S rRNA 1-butanol illsolate Phylotype No. Sequence at 37° C. PN0110 Enterococcus PTA- SEQ ID 1.7 faecium 7478 NO: 41

Example 2 Isolation of Butanol Tolerant Bacterial Strains Using Continuous Culture

The purpose of this Example was to isolate butanol tolerant bacterial strains. Environmental samples were obtained from several wastewater treatment sites and were grown in the presence of 1-butanol in continuous culture in a chemostat bioreactor. Several 1-butanol tolerant bacterial strains were isolated and identified as different species of Enterococcus.

An Appilikon Fermentor (Appilikon Inc., Clinton, N.J.) was operated as an anaerobic chemostat. This bioreactor system was composed of a 1-L dished bottom reactor, Controller ADI 1032 P100, and stirrer unit with marine and turbine impellers. Bio Controller ADI 1030 Z510300020 with appropriate sensors was used to monitor pH, dissolved oxygen, and temperature. A Cole Parmer pump and pump head (Cole Parmer Instrument Co., Vernon Hills, Ill.) were used for addition of acid and base to maintain the culture medium at pH 7.0. The temperature was maintained at 37° C. using a circulating water bath. The culture medium (S20 medium) consisted of 5 mM potassium phosphate buffer, pH 7.0, 10 mM ammonium sulfate, 0.1% yeast extract, 0.1% caseamino acids, 100 mM MOPS, 2 mM MgCl₂, 0.7 mM CaCl₂, 0.05 mM MnCl₂, 0.001 mM ZnCl₂, 0.002 mM thiamine hydrochloride, 1.72 μM CuCl₂, 2.53 μM CoCl₂, 2.42 μM Na₂MoO₄, 25 mM glucose, 12.5 mM sucrose, and 12.5 mM fructose, and a volume of 500 mL of this medium was used in the bioreactor. The bioreactor was operated with a feed rate in the range of 0.1 to 1.0 mL/minute and a stirrer speed of 50 rpm.

The bioreactor was inoculated with a mixture of several wastewater sludge samples obtained from different wastewater treatment facilities at several E.I. du Pont de Nemours and Company sites. After a short period of batch mode operation, the bioreactor was operated in continuous feed mode. 1-Butanol was added to the bioreactor as part of the influent culture medium. The level of 1-butanol in the bioreactor was 0% when the bioreactor was switched from batch mode to continuous feed mode and then 1-butanol was gradually increased to 2.5% in the bioreactor. The flow rate of the medium was in the range of 0.1 to 1.0 mL/min.

Cell density in the bioreactor was monitored by measuring the optical density at 600 nm. The 1-butanol in the feed and effluent was determined by GCMS using an HP6890 Gas Chromatograph with 5973 Mass Detector (Agilent Technologies, Inc, Wilmington, Del.). The GC column was a DB-WAX, 30 m×0.32 mm ID×0.25 μm column (J&W Scientific, Inc., Folsom, Calif.). Alternatively, samples were filtered (Acrodisc CR PTFE 0.2 μm filters) and analyzed by HPLC using a Shodex® SH1011 column (8 mm ID×300 mm length; Shoko America Inc., Colorado Springs, Colo.) with a Shodex® SH-G guard column. The mobile phase was 0.01 N sulfuric acid. The column temperature was 50° C. and a flow rate of 0.5 mL/min was used. For detection, a photometric detector at 210 nm and a refractive index detector were used. The sample injection volume was 10 μL.

After an initial adjustment period, the amount of 1-butanol entering the bioreactor through the feed was gradually increased to reach a final 1-butanol concentration in the culture medium of 2.5% w/v. During this same period, the amount of glucose in the bioreactor effluent was monitored. Increasing the amount of 1-butanol in the feed resulted in a decrease in cell density and a concomitant decrease in glucose utilization. Continued incubation resulted in the cell density and glucose utilization again increasing after adaptation to a higher level of 1-butanol. For example, increasing 1-butanol to 1.6% resulted in the cell density decreasing to less than 1.5 OD₆₀₀ with a corresponding decrease in glucose utilization. However, continued incubation resulted in the cell density increasing to 2.3 OD₆₀₀ with a corresponding increase in glucose consumption.

Phylogenetic characterization of the microbial consortium in the anaerobic bioreactor and isolation of pure strains of 1-butanol resistant bacteria from this bioreactor were performed as follows. Samples of cells taken from the bioreactor at various times and from the bioreactor waste jug were serially diluted, and the serial dilutions were plated on S20 agar medium (S20 medium with 1.6% DifCo™ Agar, Noble-Solidifying agent) without 1-butanol. Colonies were then inoculated from the S20 agar medium into 1.2 mL of S20 medium without 1-butanol in the wells of a square-well microtiter plate (Beckman Coulter Inc, Fullerton, Calif.; Catalog No. 069681). The square-well microtiter plate was sealed with an adhesive cover (Beckman Coulter Inc.; Catalog No. 538619) and incubated at 37° C. with shaking for up to 72 h. The square-well microtiter plate was used to make a master plate by dispensing 200 μL of culture from each square well into a corresponding well in a “U-bottom” microtiter plate (VWR Scientific Products, West Chester, Pa.; Catalog No. 62409-052). The cells in each well were then subcultured into 200 μL of S20 medium in the wells of U-bottom microtiter plates (BD Diagnostic Systems; Catalog No. 353077) with and without 1-butanol to identify 1-butanol-tolerant isolates. The isolates were challenged separately with 2.0% and 3.0% w/v of 1-butanol. Butanol tolerance was determined by growth in the presence of 1-butanol as determined by measuring increases in optical density at 595 nm (OD₅₉₅) on a microtiter plate reader (HTS 7000 Plus Bio Assay Reader, PerkinElmer Life and Analytical Sciences Inc., Wellesley, Mass.). Isolates that had OD₅₉₅ readings in the range of 0.1 to 0.8 after growth for 48 h at 37° C. with shaking were selected for further tolerance testing. Alternatively, isolates from the master plate were replica-plated onto S20 agar containing up to 3.4% 1-butanol using the Nunc-TSP transferable solid phase screening system (Nalgene Nunc International, Napersville, Ill.; Catalog No. 445-497). Tolerant isolates were identified by growth at 37° C. after 24 to 72 h. Several isolates, including those listed in Table 5, displayed growth in 2.5% or higher 1-butanol on a solid medium. The IC₅₀ values of the isolated strains were determined at 37° C., for 1-butanol, 2-butanol, isobutanol, 2-butanone (also called methyl ethyl ketone (MEK)) and ethanol, following the method described in Example 1. The results are summarized in Table 5. Based on the IC₅₀ values determined for each compound and a correlation seen between tolerance to 1-btuanol and to each of the other tested compounds, the identified tolerant strains are expected to grow on solid medium containing 3.9% w/v 2-butanol, 2.7% w/v isobutanol, 5.0% w/v 2-butanone, or 9.0% w/v ethanol.

The isolates were identified by sequencing the 16S rRNA genes, as described in Example 1. The SEQ ID for each isolate's 16S rRNA, the phylotype, and the isolate designations are also given in Table 5.

TABLE 5 Butanol Tolerant Bacterial Strains Isolated from Environmental Samples using Continuous Culture IC₅₀ (%) IC₅₀ (%) IC₅₀ (%) IC₅₀ (%) 16S rRNA 1- 2- iso- 2-buta- IC₅₀ (%) Isolate Phylotype Sequence butanol butanol butanol none Ethanol PN0048 Enterococcus SEQ ID 1.7 2.6 1.8 3.6 4.4 gallinarium NO: 42 PN0197- Enterococcus SEQ ID 1.8 2.9 1.8 3.2 4.1 10 faecalis NO: 43 PN0133 Enterococcus SEQ ID 1.9 2.8 2.1 3.0 4.4 faecium NO: 44 

1. An isolated butanol tolerant Enterococcus having the following characteristics: i) is a facultative anaerobe; and ii) is Gram-positive; and iii) is non-motile; and iv) has spherical or ovoid cell morphology; and v) has ability to initiate growth in a broth comprising 6.5% sodium chloride; and vi) has the ability to initiate growth in broth at pH 9.6; and vii) is tolerant to at least 2.5% w/v butanol when grown on a solid medium, wherein the butanol tolerant Enterococcus comprises a 16S rRNA gene having a sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44.
 2. The isolated butanol tolerant Enterococcus of claim 1 for use in the production of butanol. 